1. Technical Field
The present invention relates to methods for the production of virus for vaccine production in cell culture.
2. Background
Effective control of influenza pandemics depends on early vaccination with the inactivated virus produced from newly identified influenza strains. However, for more effective pandemic control, improvements in the manufacturing and testing of the vaccine are needed. Influenza viruses undergo very frequent mutations of the surface antigens. Consequently, vaccine manufacturers cannot stock-pile millions of doses for epidemic use. Current influenza control methods demand constant international surveillance and identification of any newly emergent strains coupled with vaccine production specific for the newly identified strains. Current influenza vaccine production, which requires the use of embryonated eggs for virus inoculation and incubation, is cumbersome and expensive. It can also be limited by seasonal fluctuations in the supply of suitable quality eggs. Thus, for production of mass doses of monovalent vaccine in a short time, it would be advantageous to develop alternate, egg-independent production technology. In this respect, production of an influenza vaccine on a stable cell line may solve many of the problems in mass production. However, the yield of human influenza viruses on tissue culture is disappointingly much lower than in embryonated eggs (Tannock et al. Vaccine 1985 3:333-339). To overcome these limitations and improve the quality of vaccines, it would be advantageous to develop cell culture lines which provide an enhanced yield of virus over those currently available.
In using mammalian cell lines for whole virion vaccine production, a common problem for vaccine manufacturers is that mammalian cells have intrinsic antiviral properties, specifically, the interferon (IFN) system, which interferes with viral replication. IFNs can be classified into two major groups based on their primary sequence. Type I interferons, IFN-xcex1 and IFN-xcex2, are encoded by a super family of intronless genes consisting of the IFN-xcex1 gene family and a single IFN-xcex2 gene. Type II interferon, or IFN-xcex3, consists of only a single type and is restricted to lymphocytes (T-cells and natural killer cells). Type I interferons mediate diverse biological processes including induction of antiviral activities, regulation of cellular growth and differentiation, and modulation of immune functions (Sen, G. C. and Lengyel, P. (1992) J. Biol. Chem. 267, 5017-5020; Pestka, S. and Langer, J. A. (1987) Ann. Rev. Biochem. 56, 727-777). The induced expression of Type I IFNs, which include the IFN-xcex1 and IFN-xcex2 gene families, is detected typically following viral infections. Many studies have identified promoter elements and transcription factors involved in regulating the expression of Type I IFNs (Du, W., Thanos, D. and Maniatis, T. (1993) Cell 74, 887-898; Matsuyama, T., Kimura, T., Kitagawa, M., Pfeffer, K., Kawakami, T., Watanabe, N., Kundig, T. M., Amakawa, R., Kishihara, K., Wakeham, A., Potter, J., Furlonger, C. L., Narendran, A., Suzuki, H., Ohashi, P. S., Paige, C. J., Taniguchi, T. and Mak, T. W. (1993) Cell 75, 83-97; Tanaka, N. and Taniguchi, T. (1992) Adv. Immunol. 52, 263-81). However, it remains unclear what are the particular biochemical cues that signify viral infections to the cell and the signaling mechanisms involved (for a recent review of the interferon system see Jaramillo et al. Cancer Investigation 1995 13:327-337).
IFNs belong to a class of negative growth factors having the ability to inhibit growth of a wide variety of cells with both normal and transformed phenotypes. IFN therapy has been shown to be beneficial in the treatment of human malignancies such as Kaposi""s sarcoma, chronic myelogenous leukemia, non-Hodgkin""s lymphoma and hairy cell leukemia as well as the treatment of infectious diseases such as papilloma virus (genital warts) and hepatitis B and C (reviewed by Gutterman Proc. Natl Acad Sci. 91:1198-1205 1994). Recently, genetically-engineered bacterially-produced IFN-xcex2 was approved for treatment of multiple sclerosis, a relatively common neurological disease affecting at least 250,000 patients in the U.S. alone.
IFNs elicit their biological activities by binding to their cognate receptors followed by signal transduction leading to induction of IFN-stimulated genes (ISG). Several of them have been characterized and their biological activities examined. The best studied examples of ISGs include a double-stranded RNA (dsRNA) dependent kinase (PKR, formerly known as p68 kinase), 2xe2x80x2-5xe2x80x2-linked oligoadenylate (2-5A) synthetase, and Mx proteins (Taylor J L, Grossberg S E. Virus Research 1990 15:1-26.; Williams B R G. Eur. J. Biochem. 1991 200:1-11). Human Mx A protein is a 76 kD protein that inhibits multiplication of influenza virus and vesicular stomatitis virus (Pavlovic et al. (1990) J. Viol. 64, 3370-3375).
2xe2x80x2-5xe2x80x2 Oligoadenylate synthetase (2-5A synthetase) uses ATP to synthesize short oligomers of up to 12 adenylate residues linked by 2xe2x80x2-5xe2x80x2-phosphodiester bonds. The resulting oligoadenylate molecules allosterically activate a latent ribonuclease, RNase L, that degrades viral and cellular RNAs. The 2-5A synthetase pathway appears to be important for the reduced synthesis of viral proteins in cell-free protein-synthesizing systems isolated from IFN-treated cells and presumably for resistance to viral infection in vivo at least for some classes of virus.
PKR (short for protein kinase RNA-dependent) is the only identified double-stranded RNA (dsRNA)-binding protein known to possess a kinase activity. PKR is a serine/threonine kinase whose enzymatic activation requires binding to dsRNA or to single-stranded RNA presenting internal dsRNA structures, and consequent autophosphorylation (Galabru, J. and Hovanessian, A. (1987) J. Biol. Chem. 262, 15538-15544; Meurs, E., Chong, K., Galabru, J., Thomas, N. S., Kerr, I. M., Williams, B. R. G. and Hovanessian, A. G. (1990) Cell 62, 379-390). PKR has also been referred to in the literature as dsRNA-activated protein kinase, P1/e1F2 kinase, DAI or dsI for dsRNA-activated inhibitor, and p68 (human) or p65 (murine) kinase. Analogous enzymes have been described in rabbit reticulocytes, different murine tissues, and human peripheral blood mononuclear cells (Farrel et al. (1977) Cell 11, 187-200; Levin et al. (1978) Proc. Natl Acad. Sci. USA 75, 1121-1125; Hovanessian (1980) Biochimie 62, 775-778; Krust et al. (1982) Virology 120, 240-246; Buffet-Janvresse et al. (1986) J. Interferon Res. 6, 85-96). The best characterized in vivo substrate of PKR is the alpha subunit of eukaryotic initiation factor-2 (eIF-2a) which, once phosphorylated, leads ultimately to inhibition of cellular and viral protein synthesis (Hershey, J. W. B. (1991) Ann. Rev. Biochem. 60, 717-755). PKR can phosphorylate initiation factor e1F-2xcex1 in vitro when activated by double-stranded RNA (Chong et al. (1992) EMBO J. 11, 1553-1562). This particular function of PKR has been suggested as one of the mechanisms responsible for mediating the antiviral and antiproliferative activities of IFN-xcex1 and IFN-xcex2. An additional biological function for PKR is its putative role as a signal transducer. Kumar et al. demonstrated that PKR can phosphorylate IxcexaBxcex1, resulting in the release and activation of nuclear factor xcexaB (NF-xcexaB) (Kumar, A., Haque, J., Lacoste, J., Hiscott, J. and Williams, B. R. G. (1994) Proc. Natl. Acad. Sci. USA 91, 6288-6292). Given the well-characterized NF-xcexaB site in the IFN-xcex2 promoter, this may represent a mechanism through which PKR mediates dsRNA activation of IFN-xcex2 transcription (Visvanathan, K. V. and Goodbourne, S. (1989) EMBO J. 8, 1129-1138).
The catalytic kinase subdomain of PKR (i.e., of p68 (human) kinase and p65 (murine) kinase) has strong sequence identity (38%) with the yeast GCN2 kinase (Chong et al. (1992) EMBO J. 11, 1553-1562; Feng et al. (1992) Proc. Natl. Acad. Sci. USA 89, 5447-5451). Recombinant p68 kinase expressed in yeast Saccharomyces cerevisiae exhibits a growth-suppressive phenotype. This is thought to be attributed to the activation of the p68 kinase and subsequent phosphorylation of the yeast equivalent of mammalian e1F2xcex1 (Chong et al.; Cigan et al. (1982) Proc. Natl. Acad. Sci. USA 86, 2784-2788).
The present inventor has surprisingly discovered by manipulating the expression of certain ISGs that manipulation of ISGs can have beneficial uses. They have discovered that suppression of the expression of the PKR protein or the 2-5A synthetase protein or both results in a substantially higher viral yield from virus-infected cells which is useful for enhancing the production of vaccines in animal cell culture.
A common approach to examine the biological role of PKR involves the generation of mutants deficient in the kinase activities. Since PKR possesses a regulatory site for dsRNA binding and a catalytic site for kinase activity, investigators have used block deletion or site-directed mutagenesis to generate mutants at the regulatory or catalytic site. A PKR dominant negative mutant, [Arg296]PKR, which contains a single amino acid substitution of arginine for the invariant lysine in the catalytic domain II at position 296 has been described (Visvanathan, K. V. and Goodbourne, S. (1989) EMBO J. 8, 1129-1138; D""Addario, M., Roulston, A., Wainberg, M. A. and Hiscott, J. (1990) J. Virol. 64, 6080-6089). This mutant protein [Arg296]PKR can specifically suppress the activity of endogenous wild-type PKR in vivo. Additional mutants have been generated by altering the dsRNA binding motifs. For example, Feng et al. (Proc Natl Acad Sci USA 1992 89:5447-5451) abolished dsRNA binding ability of PKR by deletional analysis to obtain mutants with deletions between amino acid residues 39-50 or 58-69. Similarly, other investigators have mutated amino acid residues in the N-terminal region to suppress dsRNA binding ability leading to loss of PKR enzymatic activities (Green S R, Mathews M B. Genes and Development 1992 6:2478-2490; McCormack S J, Ortega L G, Doohan J P, Samuels C E. Virology 1994 198:92-99). A recent article has further identified two amino acid residues that are absolutely required for dsRNA binding, namely glycine 57 and lysine 60 (McMillan N A J, Carpick B W, Hollis B, Toone W M, Zamanian-Daryoush, and Williams B R G. J. Biol. Chem. 1995 270:2601-2606). Mutants in these positions were shown to be unable to bind dsRNA in vitro and possessed no antiproliferative activity in vivo when expressed in murine macrophage cells.
The physiological significance of the loss of PKR activity in vivo has been examined in animals. Catalytically inactive PKR mutants (including [Arg296]PKR) when transfected into NIH 3T3 (mouse fibroblast) cells caused suppression of endogenous PKR activity in the transfectants. When administered to nude mice, these transfected cells caused tumor formation suggesting a tumor suppressor activity for PKR (Koromilas, A. E., Roy, S., Barber, G. N., Katze, M. G. and Sonenberg, N. (1992) Science 257, 1685-1689; Meurs, E. F., Galabru, J., Barber, G. N., Katze, M. G. and Hovanessian, A. G. (1993) Proc. Natl. Acad. Sci. USA 90, 232-236). Meurs et al. (J. Virol. 1992 66:5805) produced stable transfectants of NIH 3T3 cells with either a wild type (wt) PKR gene or a dominant negative mutant under control of a CMV promoter and showed that only transfectants receiving the wt clone were partially resistant to infection with encephalomyocarditis virus (EMCV). Lee et al. (Virol. 1993 193:1037) constructed a recombinant vaccinia virus vector containing the PKR gene under control of an inducible promoter and showed that in HeLa cells infected with the recombinant virus and induced resulted in an inhibition of the vaccinia virus protein and an overall decrease in viral yield. Henry et al. (J. Biol. Regulators and Homeostatic Agents 1994 8:15) showed that reoviral mRNAs containing a PKR activator sequence are poorly expressed in comparison with other reoviral mRNAs but that addition of 2-aminopurine, a PKR inhibitor, or transfection with a dominant negative PKR mutant, specifically increased the expression of mRNA containing the activator sequence. Maran et al. (Science 1994 265:789) showed that HeLa cells that were selectively deleted for PKR mRNA by treatment with PKR antisense oligos linked to 2xe2x80x2-5xe2x80x2 oligoA were unresponsive to activation of nuclear factor-xcexaB by the dsRNA poly(I):poly(C).
Several strategies have been utilized in the effort to improve the yield of virus obtained from cell culture for vaccine production. Different cell types have been tested to obtain the best cell line for optimum growth of specific viruses. The diploid human embryonic lung cell lines, MRC-5 and WI-38, have been developed specifically for vaccine production (see Pearson Devel. Biol. Standard. 1992 76:13-17; MacDonald, C. Critical Reviews Biotech. 1990 10:155-178; Wood et al. Biologicals 1990 18:143-146). Other attempts to improve vaccine production from cell culture include use of a low protein serum replacement factor (Candal et al. Biologicals 1991 19:213-218), and treatment of the cell culture with proteolytic enzymes (U.S. Pat. No. RE 33,164).
It is an object of the present invention to provide a method for enhanced vaccine production in cell culture. It is another object of the invention to provide methods for the evaluation of antiviral compounds and for the identification and culture of viral pathogens.
These objects are generally accomplished by providing animal cell cultures in which the expression of the interferon genes is substantially decreased from the normal level of expression. This may be effected by manipulating the level of expression of factors that function in vivo to regulate the interferon level, including interferon transcriptional regulators (for example, IRF1), interferon receptors and interferon stimulated gene products (for example PKR and 2-5A synthetase).
These objects are particularly accomplished by providing various methods using animal cell cultures in which the level of interferon-mediated antiviral protein activity, particularly for double-stranded RNA dependent kinase (PKR) and 2xe2x80x2-5xe2x80x2 Oligoadenylate synthetase (2-5A synthetase), is significantly decreased from the normal levels. Among the various methods provided are methods for vaccine production, methods for determining the antiviral activity of a compound, and methods for detecting a virus in a sample.